Immunogenic HPV L2-containing VLPs and related compositions, constructs, and therapeutic methods

ABSTRACT

The invention provides immunotherapeutic and prophylactic bacteriophage viral-like particle (VLPs) which are useful in the treatment and prevention of human papillomavirus (HPV) infections and related disorders, including cervical cancer and persistent infections associated with HPV. Related compositions (e.g. vaccines), nucleic acid constructs, and therapeutic methods are also provided. VLPs and related compositions of the invention induce high titer antibody responses against HPV L2 and protect against HPV challenge in vivo. VLPs, VLP-containing compositions, and therapeutic methods of the invention induce an immunogenic response against HPV infection, confer immunity against HPV infection, protect against HPV infection, and reduce the likelihood of infection by HPV infection.

CLAIM OF PRIORITY AND GOVERNMENT INTEREST

This application is a United States national stage application basedupon and claims priority from International Patent Application No.PCT/US2011/024030 filed Feb. 8, 2011 entitled “Immunogenic HPVL2-Containing VLPs and Related Compositions, Constructs, and TherapeuticMethods”, said application claims the benefit of U.S. provisionalapplications 61/302,836, filed Feb. 9, 2010, of same title, and61/334,826, filed May 14, 2010, of same title, the entire contents ofwhich applications are incorporated by reference herein.

This patent application was supported by NIH Grant Nos. U19-AI08408 andGM042901. The Government has certain rights in the invention.

FIELD OF THE INVENTION

In one aspect, the invention provides immunogenic HPV L2-containingviral-like particles (VLPs). Related compositions (e.g. vaccines),nucleic acid constructs, and therapeutic methods are also provided.

In one aspect, the invention relates generally to virus-like particlesand, more specifically, to a platform for peptide display based on VLPsof the RNA bacteriophage PP7.

In certain aspects, the VLPs are comprised of a coat polypeptide of thebacteriophages PP7 or MS2, wherein the coat protein is modified byinsertion of peptide antigens derived from HPV L2, the recombinant coatprotein is expressed to produce and VLP wherein the HPV L2 peptide isdisplayed on the surface of the VLP.

Immunogenic VLPs and related compositions of the invention induce hightiter antibody responses against HPV L2 and protect against HPVchallenge in an in vivo animal infection model.

BACKGROUND OF THE INVENTION

Human Papillomavirus (HPV) is the most common sexually transmittedinfectious agent worldwide and also is estimated to cause ˜500,000 casesof cancer a year (34). HPV infection is also associated with a varietyof other diseases, including cutaneous and genital warts. Over 100different HPV types have been identified, but the most commonHPV-associated cancer, cervical cancer, is associated with infection byone of a subset of about 15-18 HPVs termed “high-risk” types. Two of thehigh-risk types, HPV16 and HPV18, account for approximately 70% of allcases of cervical cancer, and six other high-risk types (types 45, 31,33, 52, 58, and 35) account for an additional 25% of all cases (33).

Conventional approaches for developing an HPV vaccine were not possiblebecause of the lack of a tissue culture system for robust HPVpropagation. However, in the early 1990s it was discovered that the L1major capsid protein of HPV could self-assemble into virus-likeparticles (VLPs) that are structurally and antigenically similar toinfectious virus (21, 24, 25, 43, 51). HPV VLPs are highly immunogenic;they induce high titer antibody responses upon vaccination of animalsand people (44). These results paved the way for the development of thetwo commercially available STI HPV vaccines. Gardasil®, developed byMerck, contains L1-VLPs derived from two high-risk HPV types (16 and 18)and two low-risk HPVs associated with genital warts (6 and 11).Cervarix®, developed by GlaxoSmithKline, contains HPV16 and 18 L1-VLPs.These vaccines have excellent safety profiles, are highly effective atpreventing infection and disease, and appear to induce long-lastingantibody responses (26).

Although some cross-reactivity has been observed between closely relatedHPV genotypes (46), the protection provided upon vaccination withL1-VLPs is largely HPV type-specific. For example, women who arevaccinated with HPV16 L1-VLPs are completely protected againstHPV16-related disease (cervical intraepithelial neoplasia; CIN), but notagainst disease caused by other high-risk HPV types (26, 31). Thetype-specific nature of neutralizing antibodies induced by HPV16 L1-VLPshas also been confirmed using in vitro neutralization assays (35, 42).Taking into account the types covered by the current vaccine, and thepossibility of some partial cross-protection, it is estimated that withcomplete vaccine coverage Gardasil® and Cervarix® could provideprotection against ˜70-80% of cervical cancers. Yet, this figure may bean overestimate; ˜50% of infected women are infected with multiplecarcinogenic types. Because infection with HPV16 and 18 can cause cancermore rapidly than other high-risk types, co-infection with HPV16 andHPV18 may mask the true risk of cancer caused by other HPV types.Because vaccinated populations are still at risk for cancer the AmericanCancer Society has recommended that vaccinated women continue to besubjected to Pap screening on an annual basis, at an annual cost of $4-5billion in the United States alone.

Inducing Broadly Neutralizing Antibodies Against HPV by Targeting theMinor Capsid Protein, L2.

Papillomaviruses encode two capsid proteins, L1 and L2. Upon expression,the major capsid protein, L1, can spontaneously self-assemble intopentamers that further assemble into VLPs, comprised of 360 copies ofL1. The minor capsid protein, L2, is not required for VLP formation, butis required for formation of infectious virions (and pseudovirions). Upto 72 copies of L2 can be incorporated in a VLP, and viral infectivitycorrelates with L2 content (5).

Although neither natural infection nor immunization with L1/L2 VLPselicits anti-L2 antibody responses, vaccination with bacteriallyexpressed L2 protein, or peptides derived from L2, results in theproduction of neutralizing antibodies that are protective in animalmodels (1, 7, 13, 19, 30). The somewhat contradictory data indicatingthat L2 is poorly immunogenic, yet is the target of highly neutralizingantibodies, can be explained by recent studies that shed light on therole of L2 during viral infection. Structural studies have shown that L2is poorly exposed on the surface of virions (5). However, it has beenproposed that after the virus binds to its primary cellular receptor thecapsid undergoes a conformational change that exposes the amino terminusof L2 (15, 45). Once exposed, 12 amino acids at the N-terminus of L2 arecleaved by a cellular protein, furin, exposing one or more L2neutralizing epitopes, and, it is theorized, allow virions to interactwith a cellular coreceptor (15, 39).

Because L2 neutralizing epitopes are not exposed until after HPVbinding, normal infection fails to induce anti-L2 neutralizing antibodyresponses. Thus, there has been little evolutionary pressure for L2 toundergo antigenic variation. Unlike L1-specific neutralizing antibodies,L2-specific neutralizing antibodies are broadly cross-neutralizing (41),suggesting that neutralizing epitopes on L2 are conserved across HPVsand even papillomaviruses (PVs) that infect different species. Forexample, antisera raised against a peptide representing amino acids 1-88from bovine papillomavirus type 1 (BPV-1) L2 can cross-neutralize adiverse panel of mucosal and cutaneous HPVs (36). Similarly, vaccinationwith HPV16 and BPV-1 L2 peptides protects rabbits against challenge withtwo different rabbit papillomaviruses (19).

In principle, L2 vaccines may be able to overcome the high productioncost and type-specific limitations of L1-VLP vaccines. Unfortunately,however, the neutralizing titers produced upon L2 vaccination areconsiderably lower than for L1 VLP vaccines, particularly againstheterologous HPV types (41). Therefore, it is likely that an L2 vaccinewill only be effective if its immunogenicity is enhanced.

VLPs Induce Strong Antibody Responses.

Virus-like particles (VLPs) make excellent vaccines. They arenon-infectious, often easier to produce than actual viruses, and,because the regularity of their capsid structure presents viral epitopesas dense, highly repetitive arrays that strongly stimulate B cells, theyare highly immunogenic. VLPs are comprised of one or more proteinsarranged geometrically into dense, repetitive arrays. These structuresare largely unique to microbial antigens, and the mammalian immunesystem has apparently evolved to respond vigorously to this arrangementof antigens. B cells specifically recognize and respond strongly to theordered array of densely spaced repetitive elements characteristic ofvirus surfaces (2, 18). Highly repetitive antigens provokeoligomerization of the membrane-associated immunoglobulin (Ig) moleculesthat constitute the B cell receptor (BCR) (3). There is evidence thatthe Ig crosslinking mediated by multivalent antigens leads to theformation of highly stable BCR-signaling microdomains that areassociated with increased signaling to the B cell (48). This signalingstimulates B cell proliferation, migration, and upregulation of bothmajor histocompatibility complex (MHC) class II and the co-stimulatorymolecules that permit subsequent interactions with T helper cells thatare required to trigger IgG secretion, affinity maturation, and thegeneration of long-lived memory B cells (9). Consequently, we and othershave shown that multivalent antigens such as VLPs can activate B cellsat much lower concentrations than monomeric antigens (4, 16, 17, 32).Hence, VLPs are innately immunogenic: they induce high titer and longlasting antibody responses at low doses, often without requiringadjuvants (22, 50).

VLPs as Flexible Platforms for Vaccine Development.

VLPs can be used as the basis for vaccines targeting the virus fromwhich they were derived (the Hepatitis B virus vaccine andaforementioned HPV vaccine are two clinically approved VLP vaccines,other VLP vaccines are in clinical trials). However, they also can beused as platforms to display practically any epitope in a highlyimmunogenic, multivalent format. Heterologous antigens displayed at highdensity on the surface of VLPs exhibit the same high immunogenicity asunmodified VLPs. VLPs derived from a variety of different viruses havebeen exploited in this manner to induce antibody responses againstheterologous targets that are poorly immunogenic in their nativecontexts. Although the VLP platform strategy has typically been appliedto target antigens derived from pathogens, VLP-display can effectivelyinduce antibody responses against practically any antigen. One exampleis the vaccine for nicotine addiction (designed to assist smokers whoare trying to quit) developed by a biotechnology company, CytosBiotechnology. This vaccine consists of nicotine, conjugated at highcopy number to the surface of VLPs derived from a bacteriophage. Inphase II clinical trials, VLPs displaying nicotine were well-toleratedand induced strong nicotine-specific IgG responses in 100% of immunizedsubjects (14). Even self-antigens, which are normally subject to themechanisms of B cell tolerance, are immunogenic when displayed at highdensity on the surface of VLPs. Vaccines have been developed againstself-molecules involved in several different diseases, includingamyloid-beta (Alzheimers (12, 27)), TNF-α (arthritis (10)), CCR5 (HIVinfection (8, 11)), gastrin (cancer, unpublished data), IgE (allergy,unpublished data), and others. VLP-based vaccines developed bypharmaceutical companies targeting amyloid-beta and angiotensin II(hypertension) are currently being evaluated in clinical trials;positive results from the trial of vaccine targeting angiotensin II (asa vaccine for hypertension) were reported in the spring of 2008 (49).

HPV vaccines that target the L2 protein have been described in a varietyof other journal articles, patents, and patent applications.

For example, Kawana et al. describe a peptide representing amino acids108-120 from HPV16 L2 that induces neutralizing antibodies effectiveagainst HPV16 and HPV11 (23). The information obtained from theseresults was said to be useful for developing a prophylactic peptidevaccine that prevents infection with genital HPVs in humans.

U.S. Pat. No. 6,174,532 and PCT/US2006/003601 described the use of theN-terminal portion of papillomavirus L2 protein or a prophylacticallyeffective peptide fragment thereof (or a prophylactically effectivepeptide derivative sequence thereof) in the production of a medicamentsuitable for use as a prophylactic agent against papillomavirusinfection in mammals.

U.S. Patent Application Document No. 2008/0213293 describes treating orpreventing respiratory papillomatosis by immunizing either a mother orchild before, during or after delivery. Immunity may be induced with avaccine comprising a HPV peptide antigen fused to a viral protein orother antigen. Antibodies and cells may be recovered from an animalpreviously vaccinated with the same vaccine. Of particular interest isthe use of HPV L2 peptides designating a neutralizing epitope of HPV.

PCT/US2008/053498 described the use of papillomavirus L2 polypeptidesproduced in a plant expression system as a prophylactic HPV vaccine.

PCT WO 93/00436 describes papillomavirus L2 protein for use in theproduction of a medicament for use in medicine, particularly for use inthe prophylaxis or therapy of papillomavirus tumours.

PCT 2004/052395 describes a vaccine composition comprising an HPV L2peptide in physical association with an HPV virus like particle (VLP).

PCT 2008/082719 describes a composition that includes: a papillomavirusvirus-like particle including an L1 protein or polypeptide and achimeric protein or polypeptide that contains at least a portion of anL2 protein 20 and a protein or polypeptide fragment including a firstepitope; and a DNA molecule encoding a protein or polypeptide includinga second epitope.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a virus-like particle (VLP)virus-like particle comprising a bacteriophage single chain coatpolypeptide dimer, preferably based upon a MS2 or PP7 bacteriophage anda HPV L2 peptide, wherein the HPV L2 peptide is displayed on thevirus-like particle and said VLP encapsidates bacteriophage mRNA, suchthat the composition is immunotherapeutic and prophylactic forHPV-induced disorders.

It is another object of the invention to provide nucleic acid constructswhich express a VLP comprising a bacteriophage single chain coatpolypeptide dimer, preferably based upon a MS2 or PP7 bacteriophage anda HPV L2 peptide, wherein the HPV L2 peptide is displayed on theviral-like particle and wherein said VLP encapsidates bacteriophagemRNA.

It is another object of the invention to provide a method of instillingimmunogenicity or prophylaxis to a HPV infection and/or a HPV relateddisorder in a patient at risk for such an infection or disorder.

SUMMARY OF THE INVENTION

The development and commercialization of the HPV vaccines based onL1-VLPs has been a significant public health breakthrough towards thegoal of eradicating HPV-associated cancers. However, the currentvaccines largely elicit type-specific immunity, meaning that it may beimpossible to use L1-VLPs to provide complete protection against themultiple high risk genotypes associated with cancer. Immunization withHPV L2 induces antibodies that broadly neutralize divergent HPV strains,but L2 is poorly immunogenic. As a consequence of the limitations ofprior art vaccines, therefore, new strategies for identifying antigenicepitopes and eliciting high-titer anti-L2 antibody responses are needed.

The invention provides immunotherapeutic and prophylactic bacteriophageviral-like particle (VLPs) which are useful in the treatment andprevention of human papillomavirus (HPV) infections and relateddisorders, including cervical cancer and persistent infectionsassociated with HPV. Related compositions (e.g. vaccines), nucleic acidconstructs, and therapeutic methods are also provided. VLPs and relatedcompositions of the invention induce high titer antibody responsesagainst HPV L2 and protect against HPV pseudovirus challenge in vivo.VLPs, VLP-containing compositions, and therapeutic methods of theinvention induce an immunogenic response against HPV infection, conferimmunity against HPV infection, protect against HPV infection, andreduce the likelihood of infection by and/or inhibit HPV infection.

Because antibodies that are specific for highly conserved epitopeswithin L2 are able to neutralize infection by a broad range of HPVtypes, HPV L2-targeting VLPs and related compositions (e.g. vaccines) ofthe invention provide a more comprehensive protection against infectionby multiple HPV types.

In one aspect, the invention provides a VLP comprising a bacteriophagesingle chain coat polypeptide dimer and a HPV L2 peptide, wherein theHPV L2 peptide is displayed on the VLP, and wherein the VLP isimmuno-prophylactic for HPV-induced disorders.

Certain aspects of the invention reflect that the single-chain dimer ofPP7 coat protein can tolerate the insertion of a wide variety ofpeptides, including peptides derived from the L2 protein of differentstrains of HPV or papillomavirus from a variety of non-human (animal)species as otherwise set forth herein, and is highly immunogenic.

Certain aspects of the invention also reflect that in an animal in vivochallenge model, a PP7 VLP displays a broadly cross-type neutralizingepitope from the HPV minor capsid protein L2 and induces antibodies thatprotect against homologous and heterologous HPV infection. Thesesequences preferably represent at least 5 consecutive amino acids,preferably from 5 consecutive amino acids to 30 consecutive amino acids(including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 consecutive amino acids withinthis range) at least 10 consecutive amino acids, 15 consecutive aminoacids, 20 consecutive amino acids, 25 consecutive amino acids or 30amino acids of HPV L2 peptide amino acid sequences 11-125, preferablyamino acids 14-40, 11-36, 17-36, 11-31, preferably, amino acids 17-31 ofL2 peptide of a variety of HPV strains as otherwise described herein,especially including strains 16, 45 and/or 58 as well as HPV strains 1,5, 6, 11 and 18. In the case of sequences 17-31 of papilloma virusstrains HPV1, HPV2, HPV5, HPV6, HPV8, HPV11, HPV16, HPV18, HPV31, HPV33,HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68,HPV73, CRPV and BPV1 (see FIG. 3 hereof) especially including HPVstrains 16, 45 and/or 58, as well as of HPV strains 1, 5, 6, 11 and 48,it is noted that the sequence of this region of HPV peptide L2 isrelatively conserved across diverse HPV isolates and the use of each ofthese sequences within the VLPs according to the present invention mayinstill immunogenic protection against a broad array of a variety ofstrains of HPV.

L2 peptide sequences as described above from any of HPV strains HPV1,HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV12,HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22,HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32,HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42,HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52,HPV53, HPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV60, HPV61, HPV62,HPV63, HPV64, HPV65, HPV66, HPV67, HPV68, HPV69, HPV70, HPV71, HPV72,HPV73, HPV74, HPV75, HPV76, HPV77, HPV78, HPV79, HPV80, HPV81, HPV82,HPV83, HPV84, HPV85, HPV86, HPV87, HPV88, HPV89, HPV90, HPV91, HPV92,HPV93, HPV94, HPV95, HPV96, HPV97, HPV98, HPV99, HPV100; and/or animalpapillomaviruses: bovine papillomavirus type 1 (BPV1), bovinepapillomavirus type 2 (BPV2), bovine papillomavirus type 4 (BPV4),cottontail rabbit papillomavirus (CRPV), deer papillomavirus (DPV),European elk papillomavirus (EEPV), canine oral papillomavirus (COPV),Rhesus monkey papillomavirus (RnPV) and rabbit oral papillomavirus(ROPV) may be displayed by the VLPs according to the present invention.

Representative L2 peptides for use in VLPs according to the presentinvention include the following for each of the above-referenced HPVstrains and animal PVs (note that all of the amino acid numbers listedbelow are referenced based upon the sequence of HPV 16 L2 and that foreach of the lengths of amino acids, each length within the length rangeis understood to be disclosed):

Linear peptides of lengths varying from 5-30 amino acids from anywherein L2 (from the list of HPV and animal PVs set forth above);

Linear peptides of lengths varying from 5-30 amino acids from aminoacids 1-121 of L2;

Linear peptides of lengths varying from 5-30 amino acids from aminoacids 1-88 of L2;

Linear peptides of lengths varying from 5-27 amino acids from aminoacids 14-40 of L2;

Linear peptides of varying lengths from 5-21 amino acids from aminoacids 17-36 of L2;

Linear peptides of varying lengths from 5-15 amino acids from aminoacids 17-31 of L2;

The following sequences of the above referenced PVs (or portions of atleast 5, 10 or 15 amino acids thereof):

-   -   aa 28-52;    -   aa 34-52;    -   aa 35-50;    -   aa 49-71;    -   aa 51-65;    -   aa 61-75;    -   aa 64-81;    -   aa 65-85;    -   aa 65-80.

Other L2 sequences are readily obtained from the detailed description ofthe invention.

In another aspect, the invention provides a composition comprising a VLPcomprising a bacteriophage single chain coat polypeptide dimer and a HPVL2 peptide as otherwise described above, wherein the HPV L2 peptide isdisplayed on the VLP and encapsidates bacteriophage mRNA, and whereinthe composition is immunotherapeutic and prophylactic for HPV-induceddisorders.

In another aspect, the invention provides a composition comprising a VLPdisplaying L2 peptides from two or more strains of HPV, such as aminoacids 17-31 from both HPV16 and HPV18 L2, on the same VLP, and whereinthe composition is immunotherapeutic and prophylactic for HPV-induceddisorders.

In certain aspects, the invention provides a VLP, or a compositioncomprising a VLP, wherein the VLP is made by transforming a prokaryotewith a nucleic acid construct comprising:

(a) a bacterial or bacteriophage promoter;

(b) a coding sequence of a bacteriophage single chain coat polypeptidedimer (preferably a PP7 coat polypeptide dimer) which is operablyassociated with the promoter and which is modified to contain anucleotide sequence encoding a HPV L2 peptide;

(c) a gene for resistance to an antibiotic which is operably associatedwith the promoter; and

(d) a replication origin for replication in a prokaryotic cell,

wherein the composition is immunotherapeutic and/or prophylactic forHPV-induced disorders.

In certain aspects, VLPs and VLP-containing compositions (e.g. vaccines)of the invention are comprised of VLPs comprising HPV L2 peptides fromdifferent HPV types. In other aspects, VLPs and VLP-containingcompositions of the invention comprise hybrid VLPs that display HPV L2sequences derived from multiple HPV types.

In certain aspects, the invention provides a VLP, or a compositioncomprising a VLP, wherein the VLP is made by transforming a prokaryotewith a nucleic acid construct comprising either:

(1) (a) a bacterial or bacteriophage promoter which is operablyassociated with a coding sequence of a bacteriophage (preferably PP7)single chain coat polypeptide dimer, wherein the coat polypeptide dimercoding sequence is modified to: (i) define a first restriction sitewhich is located in the downstream portion of the coat polypeptide dimercoding sequence and which is either positioned 5′ to, or located within(preferably within), the sequence which defines the coat polypeptidedimer AB loop, and (ii) contain a nucleotide sequence encoding a HPV L2peptide;(b) a second restriction site positioned 3′ to the coat polypeptidedimer coding sequence;(c) an antibiotic resistance gene which is operably associated with thepromoter; and(d) a replication origin for replication in a prokaryotic cell; or(2) (a) a bacterial or bacteriophage promoter which is operablyassociated with a coding sequence of a bacteriophage (preferably PP7),single chain coat polypeptide dimer, wherein the coat polypeptide dimercoding sequence is modified to: (i) define a first restriction sitewhich is located in the downstream portion of the coat polypeptide dimercoding sequence andwhich is either positioned 5′ to, or located within (preferably within),the sequence which defines the coat polypeptide dimer AB loop, and (ii)contain a nucleotide sequence encoding a HPV L2 peptide;(b) a second restriction site positioned 3′ to the coat polypeptidedimer coding sequence;(c) a PCR primer positioned 3′ to the second restriction site;(d) an antibiotic resistance gene which is operably associated with thepromoter; and(e) a replication origin for replication in a prokaryotic cell; or(3) (a) a bacterial or bacteriophage promoter which is operablyassociated with a coding sequence of a bacteriophage (preferably MS2)single chain coat polypeptide dimer, wherein the coat polypeptide dimercoding sequence is modified to (i) define a codon sequence positioned 5′to that portion of the sequence which defines the coat polypeptide dimerAB loop, and (ii) contain a nucleotide sequence encoding a HPV L2peptide;(b) a restriction site positioned 3′ to the coat polypeptide dimercoding sequence;(c) a PCR primer positioned 3′ to the second restriction site;(d) an antibiotic resistance gene for resistance to a first antibiotic,wherein the resistance gene is operably associated with the promoter;(e) a helper phage gene modified to contain a second antibioticresistance gene conferring resistance to a second antibiotic, and(f) a replication origin for replication in a prokaryotic cell.

In certain aspects, the coding sequence of the bacteriophage singlechain coat polypeptide dimer further comprises a transcriptionterminator positioned 5′ to the second restriction site.

In certain aspects, the invention provides a method of inoculating asubject at risk of developing a HPV-related disorder, the methodcomprising administering to the subject one or more doses of acomposition comprising a HPV L2-containing VLP as described herein. Inother aspects, the invention provides a method of treating a subject whois at risk of developing a HPV-related disorder and who has undergoneHPV seroconversion, the method comprising administering to the subjectone or more doses of a composition comprising a HPV L2-containing VLP asdescribed herein. In still other aspects, the invention provides amethod of treating a subject who has developed a HPV-related disorder,the method comprising administering to the subject one or more doses ofa composition comprising a HPV L2-containing VLP as described herein.

Previously, we described the use of VLPs of the RNA bacteriophage MS2for peptide display. By genetically fusing two copies of the MS2 coatprotein, we created a single-chain dimer with increased thermodynamicstability and vastly improved tolerance of insertions in its AB-loop(38). The MS2 coat protein dimer was widely tolerant of geneticinsertion of defined peptide sequences as well as random peptideinsertions. Recombinant MS2 VLPs elicited high titer IgG antibodiesagainst the inserted sequences. Moreover, MS2 coat protein single-chaindimers produced correctly assembled VLPs that specifically encapsidatedthe mRNA encoding their synthesis, raising the possibility that theycould be used in affinity selections protocols analogous to filamentousphage display.

Since MS2 is only one member of a large family of bacteriophages whoseindividual members share similar molecular biology, we suspected that,following similar design principles, other phage VLPs could be adaptedto this same purpose. For example, here we describe the engineering ofVLPs of PP7, a bacteriophage of Pseudomonas aeruginosa, for the purposesof peptide display.

PP7 VLPs offer several potential advantages and improvements over theMS2 VLP. First, the particles are dramatically more stablethermodynamically, because of the presence of stabilizing inter-subunitdisulfide bonds. For many practical applications, including vaccines,increased stability is a desirable trait. Second, PP7 VLPs are notcross-reactive immunologically with those of MS2. This could beimportant in applications where serial administration of VLPs may benecessary. Third, we theorized that the correct folding and assembly ofthe PP7 VLP might be more resistant to the destabilizing effects ofpeptide insertion, or that it might at least show tolerance of somepeptides not tolerated in MS2 VLPs. The single-chain dimer of PP7 coatprotein can tolerate the insertion of a wide variety of peptides, ishighly immunogenic, and packages the RNA that directs its synthesis.Moreover, we show in an in vivo challenge model that a PP7 VLPdisplaying a broadly cross-type neutralizing epitope from the HPV minorcapsid protein L2 induces antibodies that protect against homologous andheterologous HPV infection.

Thus, we describe the use of recombinant VLPs derived RNA bacteriophagesto induce high titer antibody responses against L2 that protect againstmultiple diverse HPV types.

These and other aspects of the invention are described further in theDetailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Plasmids for expression of recombinant PP7 coat protein,including pP7K, p2P7K32, pETP7K, and pET2P7K32.

FIG. 2. Nucleotide sequences of the plasmids p2P7K32 and pET2P7K32. SEQID No. 1

FIG. 3, The N-terminal sequence of the downstream copy of coat proteinencoded by p2P7K32, SEQ ID No. 2. Below this is a list of selectedforward primers used to clone the listed sequences into PP7 coat (shown5′ to 3′). A similar strategy was used to clone sequences from HPV1,5/8, 6, 11/33, 16, 18, 33, 39/45, and 52/58 L2 into the PP7 coatprotein. The KpnI restriction site is shown in italics and the peptideinsertion is shown in bold text. All of these primers yield an insertionwhich is flanked by a thr residue on the N-terminal side and a glu (thewild-type position 11 amino acid) on the C-terminal side.

FIG. 4. Amino acid sequence of L2 (17-31, or equivalent) from selectedSTI/carcinogenic/cutaneous HPV and animal papillomavirus types, adaptedfrom (1). PP7 VLPs displaying a subset of these sequences wereconstructed. The following sequence ID Nos. apply to the disclosed aminoacid sequences: HPV1 (SEQ ID No. 12), HPV5 (SEQ ID No. 13), HPV8 (SEQ IDNo. 14), HPV16 (SEQ ID No. 15), HPV35 (SEQ ID No. 16), HPV31 (SEQ ID No.17), HPV33 (SEQ ID No. 18), HPV58 (SEQ ID No. 19), HPV52 (SEQ ID No.20), HPV73 (SEQ ID No. 21), HPV6 (SEQ ID No. 22), HPV11 (SEQ ID No. 23),HPV18 (SEQ ID No. 24), HPV45 (SEQ ID No. 25), HPV39 (SEQ ID No. 26),HPV68 (SEQ ID No. 27), HPV59 (SEQ ID No. 28), HPV51 (SEQ ID No. 29),HPV56 (SEQ ID No. 30), HPV66 (SEQ ID No. 31), HPV2 (SEQ ID No. 32), CPRV(SEQ ID No. 33) and BPV1 (SEQ ID No. 34).

FIG. 5. Agarose gel electrophoresis of purified wild-type single-chaindimer (lane 1), 16L2 (lane 2), 45L2 (lane 3), and 58L2 (lane 4) VLPs.Variations in electrophoretic mobility reflect charge differencesconferred by the inserted peptides.

FIG. 6. Binding of an anti-16L2 monoclonal antibody against (RG-1) torecombinant VLPs. (A) Dilutions of the mAb was reacted with 500 ng/wellof wild-type PP7, 16L2, and Flag-VLPs. Binding was detected using ahorseradish peroxidase-labeled goat anti-mouse IgG secondary followed bydevelopment with ABTS. Reactivity was determined by measurement of theabsorbance at 405 nm (OD 405). (B) Reactivity of a 1:5000 dilution ofRG-1 mAb to the eight recombinant L2 VLPs constructed, or to wild-typePP7 VLPs.

FIG. 7. IgG antibody responses in groups of mice immunized withwild-type PP7 VLPs, 1L2-VLPs, 5L2-VLPs, 6L2-VLPs, 11L2-VLPs, 16L2-VLPs,18L2-VLPs, 45L2-VLPs, and 58L2VLPs. End-point dilution ELISA titersagainst a peptides representing amino acids 14-40 from the appropriateHPV L2 (shown in the key) conjugated to streptavidin. 10 μg of VLPs wereadministered intramuscularly in the presence of incomplete Freund'sadjuvant. Results are from sera obtained three to four weeks after thesecond vaccination. Each datum point represents the antibody titer froman individual mouse. Lines represent the geometric mean titer for eachgroup.

FIG. 8. Mice immunized with PP7 16L2-VLPs are protected from vaginalchallenge with HPV16 or HPV45 pseudovirions. Groups of five mice wereimmunized two times with 10 μg 16L2-VLPs, wild-type PP7 VLPs, or HPV16L1-VLPs formulated in incomplete Freund's adjuvant (IFA). As anadditional control, mice were immunized with IFA alone. Three weeksafter the second immunization mice were intravaginally challenged with10⁸ IU of HPV16 pseudovirus (left panel) or HPV45 pseudovirus (rightpanel) containing a luciferase reporter. As a control, a group of fivemice were not infected. Luciferase activity was quanititated 48 hoursafter infection by taking images 3 min post-installation of luciferin atmedium binning with a 30-s exposure. Images were then analyzed bydrawing an equally sized region of interest for each mouse and measuringaverage radiance (photons/second/cm²/sr) within this region. Resultsshown are the mean average radiance for each group of five mice. Errorbars represent the standard error of the mean. Lines above pairs of dataindicate the percent reduction of signal in mice immunized with16L2-VLPs relative to wild-type PP7 VLPs or the IFA control. Allcomparisons shown here are statistically significant (p<0.01) ascalculated by T-test.

FIG. 9. Immunization with a mixture of L2-PP7 VLPs induces broad anti-L2IgG responses. Mice were immunized three times without adjuvant with 10μg (total) of a mixture of equal amounts of 1L2-VLPs, 5L2-VLPs,6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. Twoweeks after the final immunization, sera was taken as tested forreactivity to HPV L2 peptides representing L2 amino acids 14-40 fromHPV1, HPV5, HPV6, HPV16, and HPV18 sequences.

FIG. 10. Immunization with a mixture of L2-PP7 VLPs protects fromgenital infection with HPV5, HPV6, HPV16, HPV18, HPV31, HPV45, HPV52,and HPV58 pseudovirions. Groups of five mice were immunized three timeswith 10 μg (total) of a mixture of equal amounts of 1L2-VLPs, 5L2-VLPs,6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. Twoweeks after the final immunization, mice were challenged with 10⁶-10⁸ IUof the indicated pseudovirus containing a luciferase reporter. Ascontrols for each pseudovirus infection, groups of five mice were alsoimmunized with wild-type PP7 VLPs. Luciferase activity was quanititated48 hours after infection as described in FIG. 8. The extent ofprotection was determined by comparing the luciferase signal in thegroup immunized with control VLPs with the group immunized with mixedL2-VLPs.

FIG. 11. Antibody responses in mice immunized with VLPs displaying HPV16L2 amino acids 35-50 or 51-65. Mice were immunized three times and thensera was taken and tested for reactivity with synthetic peptidesrepresenting A) HPV16 L2 amino acids 34-52, and B) HPV16 L2 amino acids49-71.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal and preferablya human, to whom treatment, including prophylactic treatment(prophylaxis), with the immunogenic compositions and/or vaccinesaccording to the present invention is provided. For treatment of thoseinfections, conditions or disease states which are specific for aspecific animal such as a human patient, the term patient refers to thatspecific animal. In most instances, the patient or subject of thepresent invention is a human patient of either or both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe a number of VLP's or an amount of a VLP-containing compositionwhich, in context, is used to produce or effect an intended result,whether that result relates to the prophylaxis and/or therapy of anHPV-induced or HPV-related disorder or disease state or as otherwisedescribed herein. The term effective subsumes all other effective amountor effective concentration terms (including the term “therapeuticallyeffective”) which are otherwise described or used in the presentapplication.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded DNA and RNA. Apolynucleotide may include nucleotide sequences having differentfunctions, such as coding regions, and non-coding regions such asregulatory sequences (e.g., promoters or transcriptional terminators). Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidecan be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers (eg., dimers, tetramers). Thus, the terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring.

The term “single-chain dimer” refers to a normally dimeric protein whosetwo subunits have been genetically (chemically, through covalent bonds)fused into a single polypeptide chain. Specifically, in the presentinvention single-chain dimer versions of PP7 coat proteins wereconstructed. Each of these proteins is naturally a dimer of identicalpolypeptide chains. In the PP7 coat protein dimers the N-terminus of onesubunit lies in close physical proximity to the C-terminus of thecompanion subunit. Single-chain coat protein dimers were produced usingrecombinant DNA methods by duplicating the DNA coding sequence of thecoat proteins and then fusing them to one another in tail to headfashion. The result is a single polypeptide chain in which the coatprotein amino acid appears twice, with the C-terminus of the upstreamcopy covalently fused to the N-terminus of the downstream copy. Normally(wild-type) the two subunits are associated only through noncovalentinteractions between the two chains. In the single-chain dimer thesenoncovalent interactions are maintained, but the two subunits haveadditionally been covalently tethered to one another. This greatlystabilizes the folded structure of the protein and confers to it itshigh tolerance of peptide insertions as described above.

This application makes frequent reference to coat protein's “AB-loop”.The RNA phage coat proteins possess a conserved tertiary structure. ThePP7 coat proteins, for example, possess a structure wherein each of thepolypeptide chains is folded into of a number of β-strands. Theβ-strands A and B form a hairpin with a three-amino acid loop connectingthe two strands at the top of the hairpin, where it is exposed on thesurface of the VLP. As evidenced in the present application, peptidesinserted into the AB-loop are exposed on the surface of the VLP and arestrongly immunogenic.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional is retained by the polypeptide. NH₂ refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to thefree carboxy group present at the carboxy terminus of a polypeptide.

The term “valency” is used to describe the density of L2 peptide displayon VLPs according to the present invention. Valency in the presentinvention may range from low valency to high valency, about less than 1to more than about 180, preferably about 90 to 180. Immunogeniccompositions according to the present invention comprise VLPs which arepreferably high valency and comprise VLPs which display at least 50-60up to about 180 or more L2 peptides.

The term “coding sequence” is defined herein as a portion of a nucleicacid sequence which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′-end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′-end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

A “heterologous” region of a recombinant cell is an identifiable segmentof nucleic acid within a larger nucleic acid molecule that is not foundin association with the larger molecule in nature.

An “origin of replication”, used within context, normally refers tothose DNA sequences that participate in DNA synthesis by specifying aDNA replication initiation region. In the presence of needed factors(DNA polymerases, and the like) an origin of replication causes orfacilitates DNA associated with it to be replicated. By way of anon-limiting example, the ColE1 replication origin endows many commonlyused plasmid cloning vectors with the capacity to replicateindependently of the bacterial chromosome. Another example is the p15Areplication origin. The presence on a plasmid of an additional origin ofreplication from phage M13 confers the additional ability to replicateusing that origin when E. coli cells are infected with a so-calledhelper phage (e.g. M13CM1) which provides necessary protein factors. M13replicates intracellularly as double-stranded circular DNA, but alsoproduces a single-stranded circular form, which it packages within thephage particle. These particles provide a convenient source ofsingle-stranded circular DNA for plasmids.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence includes the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter will be found DNA sequences responsiblefor the binding of RNA polymerase and any of the associated factorsnecessary for transcription initiation. In bacteria promoters normallyconsist of −35 and −10 consensus sequences and a more or less specifictranscription initiation site. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes. Bacterial expressionvectors (usually plasmids or phages) typically utilize promoters derivedfrom natural sources, including those derived from the E. coli Lactose,Arabinose, Tryptophan, and ProB operons, as well as others frombacteriophage sources. Examples include promoters from bacteriophageslambda, T7, T3 and SP6.

In bacteria, transcription normally terminates at specific transcriptiontermination sequences, which typically are categorized as rho-dependentand rho-independent (or intrinsic) terminators, depending on whetherthey require the action of the bacterial rho-factor for their activity.These terminators specify the sites at which RNA polymerase is caused tostop its transcription activity, and thus they largely define the3′-ends of the RNAs, although sometimes subsequent action ofribonucleases further trims the RNA.

An “antibiotic resistance gene” refers to a gene that encodes a proteinthat renders a bacterium resistant to a given antibiotic. For example,the kanamycin resistance gene directs the synthesis of aphosphotransferase that modifies and inactivates the drug. The presenceon plasmids of a kanamycin resistance gene provides a mechanism toselect for the presence of the plasmid within transformed bacteria.Similarly, the chloramphenicol resistance gene allows bacteria to growin the presence of the drug by producing an acetyltransferase enzymethat inactivates the antibiotic through acetylation.

The term “PCR” refers to the polymerase chain reaction, a technique usedfor the amplification of specific DNA sequences in vitro. The term “PCRprimer” refers to DNA sequences (usually synthetic oligonucleotides)able to anneal to a target DNA, thus allowing a DNA polymerase (e.g. TaqDNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are usedin the polymerase chain reaction to initiate DNA synthesis on each ofthe two strands of a DNA and to thus amplify the DNA segment between twoprimers. Representative PCR primers which used in the present inventionare those which are presented in FIG. 3 hereof. Additional PCR primersmay be obtained for the various HPV L2 peptides which are presentedherein.

Examples of primers used for PCR are given in FIG. 3 as described aboveand the following.

E3.2: 5′ CGG GCT TTG TTA GCA GCC GG 3′—(SEQ ID No. 35) serves as the 3′(reverse)-primer in PCR reactions to amplify coat protein.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence. Transcriptional controlsequences are DNA regulatory sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide for theexpression of a coding sequence in a host cell. Translational controlsequences determine the efficiency of translation of a messenger RNA,usually by controlling the efficiency of ribosome binding andtranslation initiation. For example, as discussed elsewhere in thisapplication, the coat proteins of the RNA phages are well-knowntranslational repressors of the phage replicase. As coat proteinaccumulates to a sufficiently high concentration in the infected cell,it binds to an RNA hairpin that contains the translation initiationregion (Shine-Dalgarno and initiator AUG) of the phage's replicase gene.This prevents ribosome binding and shuts off replicase synthesis at atime in the viral life cycle where the transition from replication tovirus assembly occurs.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid, which normally replicate independently of thebacterial chromosome by virtue of the presence on the plasmid of areplication origin. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

It should be appreciated that also within the scope of the presentinvention are nucleic acid sequences encoding the polypeptide(s) of thepresent invention, which code for a polypeptide having the same aminoacid sequence as the sequences disclosed herein, but which aredegenerate to the nucleic acids disclosed herein. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid.

As used herein, “epitope” refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 5 such amino acids, and more usually, consists ofat least about 8-10 up to about 20 or more such amino acids. Methods ofdetermining the spatial conformation of amino acids are known in theart, and include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance.

As used herein, a “mimotope” is a peptide that mimics an authenticantigenic epitope.

As used herein, the term “coat protein(s)” refers to the protein(s) of abacteriophage or a RNA-phage capable of being incorporated within thecapsid assembly of the bacteriophage or the RNA-phage.

As used herein, a “coat polypeptide” as defined herein is a polypeptidefragment of the coat protein that possesses coat protein function andadditionally encompasses the full length coat protein as well orsingle-chain variants thereof.

As used herein, the term “immune response” refers to a humoral immuneresponse and/or cellular immune response leading to the activation orproliferation of B- and/or T-lymphocytes and/or antigen presentingcells. In some instances, however, the immune responses may be of lowintensity and become detectable only when using at least one substancein accordance with the invention. “Immunogenic” refers to an agent usedto stimulate the immune system of a living organism, so that one or morefunctions of the immune system are increased and directed towards theimmunogenic agent. An “immunogenic polypeptide” is a polypeptide thatelicits a cellular and/or humoral immune response, whether alone orlinked to a carrier in the presence or absence of an adjuvant.Preferably, antigen presenting cell may be activated.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal and provides ameasure of protection (protective effect) against a disease state orcondition for which the vaccine is administered. The term “prevention”or “prophylaxis” is used in context synonymously with the term “reducingthe likelihood of” or “inhibiting” wherein the measure of prevention (ofa disease state or condition) is one of degree of effect. Vaccinesaccording to the present invention may also instill immunity in apatient or subject against a disease state or condition and suchimmunity is consistent with the use of the term “prevention” or“prophylaxis” as used above.

As used herein, the term “virus-like particle of a bacteriophage” refersto a virus-like particle (VLP) resembling the structure of abacteriophage, being non-replicative and noninfectious, and lacking atleast the gene or genes encoding for the replication machinery of thebacteriophage, and typically also lacking the gene or genes encoding theprotein or proteins responsible for viral attachment to or entry intothe host.

This definition should, however, also encompass virus-like particles ofbacteriophages, in which the aforementioned gene or genes are stillpresent but inactive, and, therefore, also leading to non-replicativeand noninfectious virus-like particles of a bacteriophage.

VLP of RNA bacteriophage coat protein: The capsid structure formed fromthe self-assembly of one or more subunits of RNA bacteriophage coatprotein and optionally containing host RNA is referred to as a “VLP ofRNA bacteriophage coat protein”. In a particular embodiment, the capsidstructure is formed from the self assembly of 90-180 subunits.

A “hybrid VLP” refers to a VLP that displays two or more heterologousamino acid sequences on its surface, such as, for example, amino acids17-31 from HPV16 and HPV18. Such a hybrid VLP could be formed bycoexpression of two recombinant coat proteins in the same expressionstrain of bacteria. Alternatively, hybrid VLPs could be generated invitro by disassembly of two separate recombinant VLPs into coat proteindimers. Following disassembly, the recombinant coat protein dimers maybe mixed together and then reassembled. Methods for assembly anddisassembly of PP7 VLPs are described by Caldeira and Peabody (6).

A nucleic acid molecule is “operatively linked” to, or “operablyassociated with”, an expression control sequence when the expressioncontrol sequence controls and regulates the transcription andtranslation of nucleic acid sequence. The term “operatively linked”includes having an appropriate start signal (e.g., ATG) in front of thenucleic acid sequence to be expressed and maintaining the correctreading frame to permit expression of the nucleic acid sequence underthe control of the expression control sequence and production of thedesired product encoded by the nucleic acid sequence. If a gene that onedesires to insert into a recombinant DNA molecule does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene.

HPV-Induced Disorders, Immunogenicity, and Prophylactic Efficacy

“HPV-induced disorders” or “HPV-related disorders” include, but are notlimited to, the disorders identified in this section. Immunogenicity andprophylactic efficacy (e.g. whether a composition is immunotherapeuticand prophylactic for HPV-induced disorders) may be evaluated either bythe techniques and standards mentioned in this section, or through othermethodologies that are well-known to those of ordinary skill in the art.

Over 100 different HPV types have been identified and are referred to bynumber. Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 areamong the “high-risk” sexually transmitted HPVs and may lead to thedevelopment of cervical intraepithelial neoplasia (CIN), vulvarintraepithelial neoplasia (VIN), vaginal cancer, penile intraepithelialneoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). Severaltypes of HPV, particularly type 16, have been found to be associatedwith oropharyngeal squamous-cell carcinoma, a form of head and neckcancer. HPV (e.g., HPV 6 and 11) cause genital warts, and HPV 6 and 11can cause recurrent respiratory papillomatosis. HPV may causeepidermodysplasia verruciformis in immunocompromised individuals. OtherHPV types, such as HPV1, can causes cutaneous warts.

High-risk human HPV infection of the cervical epithelium is causallylinked with the generation of cervical cancer. HPV16 is associated withpremalignant and malignant diseases of the genito-urinary tract, and inparticular with carcinoma of the cervix. Papillomavirus prophylacticimmunogenic compositions or vaccines target the systemic immune systemfor induction of neutralizing antibodies that protect the basal cellsagainst infection. Because the carcinogenic HPVs are susceptible toneutralization by antibodies for 9-48 hours after reaching the basalcells, both low and high titered HPV type-specific antibodies induced byHPV L2-based vaccines should prove highly efficacious.

To assess immunogenicity (e.g. whether a composition has induced a hightiter antibody responses against HPV L2, an anti-HPV 16 L2 geometricmean titer (GMT) can be measured, e.g. after a few weeks of treatment(e.g. 3 or 4 weeks) and after administration of a few dosages (e.g. 3 or4). The percentage of subjects who seroconverted for HPV 16 after a fewweeks of treatment (e.g. 3 or 4 weeks) and after administration of a fewdosages (e.g. 3 or 4) and the magnitude of these responses can also bedetermined to assess immunogenicity.

HPV L2

“HPV L2” as used herein includes the L2 capsid proteins of all humanpapillomaviruses.

Production of Virus-Like Particles

The present invention is directed to virus-like phage particles as wellas methods for producing these particles in vivo or in vitro. Themethods typically include producing virus-like particles (VLPs) andrecovering the VLPs. As used herein, producing VLPs “in vitro” refers toproducing VLPs outside of a cell, for instance, in a cell-free system,while producing virions “in vivo” refers to producing VLPs inside acell, for instance, an Eschericia coli or Pseudomonas aeruginosa cell.

Bacteriophages

The system envisioned here is based on the properties of single-strandRNA bacteriophages [RNA Bacteriophages, in The Bacteriophages. Calendar,R L, ed. Oxford University Press. 2005]. The known viruses of this groupattack bacteria as diverse as E. coli, Pseudomonas and Acinetobacter.Each possesses a highly similar genome organization, replicationstrategy, and virion structure. In particular, the bacteriophagescontain a single-stranded (+)-sense RNA genome, contain maturase, coatand replicase genes, and have small (<300 angstrom) icosahedral capsids.These preferably include but are not limited to PP7, Qβ, R17, SP, PP7,GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNA bacteriophages. PP7bacteriophages are used in preferred aspects of the present invention.

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and adistant relative to coliphages like MS2 and Qβ, which also may be usedin the present invention. PP7 coat protein is a specific RNA-bindingprotein, capable of repressing the translation of sequences fused to thetranslation initiation region of PP7 replicase. Its RNA binding activityis specific since it represses the translational operator of PP7, butdoes not repress the operators of the MS2 or Qβ phages. Conditions forthe purification of coat protein and for the reconstitution of its RNAbinding activity from disaggregated virus-like particles have beenestablished. Its dissociation constant for PP7 operator RNA in vitro wasdetermined to be about 1 nM. Using a genetic system in which coatprotein represses translation of a replicase-β-galactosidase fusionprotein, amino acid residues important for binding of PP7 RNA wereidentified (28).

The coat proteins of several single-strand RNA bacteriophages are knowntranslational repressors. They shut off viral replicase synthesis bybinding an RNA hairpin that contains the replicase ribosome bindingsite. X-ray structure determination of RNA phages shows that homologiesevident from comparisons of coat protein amino acid sequences arereflected in their tertiary structures. The coat protein dimer, which isboth the repressor and the basic building block of the virus particle,consists of two intertwined monomers that together form a large β-sheetsurface upon which the RNA is bound. Each of the coat proteins uses acommon structural framework to bind different RNAs, thereby presentingan opportunity to investigate the basis of specific RNA-proteinrecognition. We have described the RNA binding properties of the coatprotein of PP7, an RNA bacteriophage of Pseudomonas aeroginosa whosecoat protein shows only 13% amino acid sequence identity to that of MS2.We have also presented the following findings. 1) The coat protein ofPP7 is a translational repressor. 2) An RNA hairpin containing the PP7replicase translation initiation site is specifically bound by PP7 coatprotein both in vivo and in vitro, indicating that this structurerepresents the translational operator. 3) The RNA binding site resideson the coat protein β-sheet.

By way of comparison, the genome of MS2 comprises a single strand of(+)-sense RNA 3569 nucleotides long, encoding only four proteins, two ofwhich are structural components of the virion. The viral particle iscomprised of an icosahedral capsid made of 180 copies of coat proteinand one molecule of maturase protein together with one molecule of theRNA genome. Coat protein is also a specific RNA binding protein.Assembly may possibly be initiated when coat protein associates with itsspecific recognition target an RNA hairpin near the 5′-end of thereplicase cistron. The virus particle is then liberated into the mediumwhen the cell bursts under the influence of the viral lysis protein. Theformation of an infectious virus requires at least three components,namely coat protein, maturase and viral genome RNA, but experiments showthat the information required for assembly of the icosahedral capsidshell is contained entirely within coat protein itself. For example,purified coat protein can form capsids in vitro in a process stimulatedby the presence of RNA [Beckett et al., 1988, J. Mol Biol 204: 939-47].Moreover, coat protein expressed in cells from a plasmid assembles intoa virus-like particle in vivo (37).

Examples of PP7 coat polypeptides include but are not limited to thevarious chains of PP7 Coat Protein Dimer in Complex With RNA Hairpin(e.g. Genbank Accession Nos. 2QUXR; 2QUXO; 2QUX_L; 2QUX_I; 2QUX_F; and2QUX_C). See also Example 1 herein and (29).

PP7 Coat Polypeptide

The coat polypeptides useful in the present invention also include thosehaving similarity with one or more of the coat polypeptide sequencesdisclosed above. The similarity is referred to as structural similarity.Structural similarity may be determined by aligning the residues of thetwo amino acid sequences (i.e., a candidate amino acid sequence and theamino acid sequence) to optimize the number of identical amino acidsalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. A candidate amino acidsequence can be isolated from a single stranded RNA virus, or can beproduced using recombinant techniques, or chemically or enzymaticallysynthesized. Preferably, two amino acid sequences are compared using theBESTFIT algorithm in the GCG package (version 1 0.2, Madison Wis.), orthe Blastp program of the BLAST 2 search algorithm available on theworldwide web at URL ncbi.nlm.nih.gov/blast/bl2seq/bl2.html. Preferably,the default values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapxdropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, a coatpolypeptide also includes polypeptides with an amino acid sequencehaving at least 80% amino acid identity, at least 85% amino acididentity, at least 90% amino acid identity, or at least 95% amino acididentity to one or more of the amino acid sequences disclosed above.Preferably, a coat polypeptide is active. Whether a coat polypeptide isactive can be determined by evaluating the ability of the polypeptide toform a capsid and package a single stranded RNA molecule. Such anevaluation can be done using an in vivo or in vitro system, and suchmethods are known in the art and routine. Alternatively, a polypeptidemay be considered to be structurally similar if it has similar threedimensional structure as the recited coat polypeptide and/or functionalactivity.

The HPV L2 peptide sequence may be present at the amino-terminal end ofa coat polypeptide, at the carboxy-terminal end of a coat polypeptide,or it may be present elsewhere within the coat polypeptide. Preferably,the HPV L2 peptide sequence is present at a location in the coatpolypeptide such that the insert sequence is expressed on the outersurface of the capsid. In a particular embodiment, the HPV L2 peptidesequence may be inserted into the AB loop regions of the above-mentionedcoat polypeptides, preferably in the downstream subunit of thesingle-chain dimer of the coat polypeptide. Examples of such locationsinclude, for instance, insertion or replacement of the insert sequenceinto a coat polypeptide in accordance with the examples presentedhereinafter. Insertion or replacement, preferably insertion, of the L2peptide sequence into the AB loop region at amino acid units 8-11 of theAB loop, preferably in the downstream subunit of the single-chain dimercoat polypeptide, is preferred.

Alternatively, the HPV L2 peptide sequence may be inserted at theN-terminus or C-terminus of the coat polypeptide, preferably in thedownstream subunit of the dimer coat polypeptide.

The HPV L2 peptide sequence preferably includes but is not limited toamino acid sequences of, at least, five, ten, fifteen, twenty amino,twenty five or thirty amino acids derived from the minor capsid proteinL2 of human Papillomavirus types 1-100, preferably 16 (HPV16), 18, 31,33, 35, 39, 45, 51, 52, 56, 58 or 59, preferably 16 (HPV16).

In another particular embodiment, the L2 peptide sequence includes aminoacid sequences with at least 75%, 80%, 85%, 90%, or 95% homology to L2sequences derived from HPV strains representing the five clades of thevirus (HPV1, HPV5, HPV6, HPV16, and HPV18).

In order to determine a corresponding position in a structurally similarcoat polypeptide, the amino acid sequence of this structurally similarcoat polypeptide is aligned with the sequence of the named coatpolypeptide as specified above.

In a particular embodiment, the coat polypeptide is a single-chain dimercontaining an upstream and downstream subunit. Each subunit contains afunctional coat polypeptide sequence. The HPV L2 peptide sequence may beinserted in the upstream and/or downstream subunit at the sitesmentioned hereinabove, e.g., AB loop region of downstream subunit,preferably at amino acid units 8-11 and as otherwise specified in theexamples which are described hereinbelow. In a particular embodiment,the coat polypeptide is a single chain dimer of a PP7 coat polypeptideand the L2 peptide sequence is inserted in the AB loop region of thedownstream subunit.

Preparation of Transcription Unit

The transcription unit of the present invention comprises an expressionregulatory region, (e.g., a promoter), a sequence encoding a singlechain of a coat polypeptide which includes a HPV L2 peptide encodingsequence and a transcription terminator. The RNA polynucleotide mayoptionally include a coat recognition site (also referred to a“packaging signal”, “translational operator sequence”, “coat recognitionsite”). Alternatively, the transcription unit may be free of thetranslational operator sequence. The promoter, coding region,transcription terminator, and, when present, the coat recognition site,are generally operably linked. “Operably linked” or “operably associatedwith” refer to a juxtaposition wherein the components so described arein a relationship permitting them to function in their intended manner.A regulatory sequence is “operably linked” to, or “operably associatedwith”, a coding region when it is joined in such a way that expressionof the coding region is achieved under conditions compatible with theregulatory sequence. The coat recognition site, when present, may be atany location within the RNA polynucleotide provided it functions in theintended manner.

The invention is not limited by the use of any particular promoter, anda wide variety of promoters are known. The promoter used in theinvention can be a constitutive or an inducible promoter. Preferredpromoters are able to drive high levels of RNA encoded by me codingregion encoding the coat polypeptide Examples of such promoters areknown in the art and include, for instance, the lac promoter, T7, T3,and SP6 promoters.

The nucleotide sequences of the coding regions encoding coatpolypeptides described herein are readily determined. These classes ofnucleotide sequences are large but finite, and the nucleotide sequenceof each member of the class can be readily determined by one skilled inthe art by reference to the standard genetic code. Furthermore, thecoding sequence of an RNA bacteriophage single chain dimer coatpolypeptide comprises a site for insertion of HPV L2 peptide-encodingsequences. In a particular embodiment, the site for insertion of the HPVL2 peptide-encoding sequence is a restriction enzyme site.

In a particular embodiment, the coding region encodes a single-chaindimer of the coat polypeptide, preferably a PP7 coat polypeptide. In amost particular embodiment, the coding region encodes a modified singlechain coat polypeptide dimer, where the modification comprises aninsertion of a coding sequence at least four amino acids at theinsertion site. The transcription unit may contain a bacterial promoter,such as a lac promoter or it may contain a bacteriophage promoter, suchas a T7 promoter and optionally a T7 transcription terminator.

In addition to containing a promoter and a coding region encoding afusion polypeptide, the RNA polynucleotide typically includes atranscription terminator, and optionally, a coat recognition site. Acoat recognition site is a nucleotide sequence that forms a hairpin whenpresent as RNA. This is also referred to in the art as a translationaloperator, a packaging signal, and an RNA binding site. Without intendingto be limiting, this structure is believed to act as the binding siterecognized by the translational repressor (e.g., the coat polypeptide),and initiate RNA packaging. The nucleotide sequences of coat recognitionsites are known in the art. Other coat recognition sequences have beencharacterized in the single stranded RNA bacteriophages R17, GA, Qβ, SP,and PP7, and are readily available to the skilled person. Essentiallyany transcriptional terminator can be used in the RNA polynucleotide,provided it functions with the promoter. Transcriptional terminators areknown to the skilled person, readily available, and routinely used.

Synthesis

As will be described in further detail below, the VLPs of the presentinvention may be produced in vivo by introducing transcription unitsinto bacteria, especially if transcription units contain a bacterialpromoter Alternatively VLPs synthesized in vitro in a coupled cell-freetranscription/translation system.

Assembly of VLPs Encapsidating Heterologous Substances

As noted above, the VLPs of the present invention display a HPV L2peptide-encoding sequence. These VLPs may be assembled by performing anin vitro VLP assembly reaction. Specifically, purified coat proteinsubunits are obtained from VLPs that have been disaggregated with adenaturant (usually acetic acid). The protein subunits are mixed with aheterologous substance. In a particular embodiment, the substance hassome affinity for the interior of the VLP and is preferably negativelycharged. This substance could include an adjuvant, including, but notlimited to RNA, bacterial DNA (CpG oligonucleotides), cholera toxinsubunit B, or E. coli lymphotoxin,

Synthesis

In a particular embodiment, the populations of the present invention maybe synthesized in a coupled in vitro transcription/translation systemusing procedures known in the art (see, for example, U.S. Pat. No.7,008,651, relevant portions of which are incorporated by referenceherein). In a particular embodiment, bacteriophage T7 (or a related) RNApolymerase is used to direct the high-level transcription of genescloned under control of a T7 promoter in systems optimized toefficiently translate the large amounts of RNA thus produced.

Uses of VLPs and VLP Populations

There are a number of possible uses for the VLPs and VLP populations ofthe present invention. As will be described in further detail below, theVLPs may be used as immunogenic compositions, particularly vaccines.

Immunogenic Compositions

As noted above, the VLPs of the present invention may be used toformulate immunogenic compositions, particularly vaccines. The vaccinesshould be in a form that is capable of being administered to an animal.Typically, the vaccine comprises a conventional saline or bufferedaqueous solution medium in which the composition of the presentinvention is suspended or dissolved. In this form, the composition ofthe present invention can be used conveniently to prevent, ameliorate,or otherwise treat a condition or disorder. Upon introduction into ahost, the vaccine is able to provoke an immune response including, butnot limited to, the production of antibodies and/or cytokines and/or theactivation of cytotoxic T cells, antigen presenting cells, helper Tcells, dendritic cells and/or other cellular responses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention. The term “adjuvant”as used herein refers to non-specific stimulators of the immune responseor substances that allow generation of a depot in the host which whencombined with the vaccine of the present invention provide for an evenmore enhanced immune response. A variety of adjuvants can be used.Examples include complete and incomplete Freund's adjuvant, aluminumhydroxide, and modified muramyl dipeptide. Squalene has also been usedas an adjuvant.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention.

EXAMPLES

The invention may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.References corresponding to numerical reference citations are listedafter the examples.

Materials and Methods

Bacteriophages PP7 and MS2.

MS2 and PP7 coat protein single-chain dimers are highly tolerant ofpeptide insertions and produce correctly assembled VLPs displaying thepeptide insertion on the surface of VLP in a highly dense, repetitivearray. These VLPs are highly immunogenic and confer this highimmunogenicity to heterologous peptides displayed on their surfaces.Here we describe VLPs displaying a peptide antigens derived from theHuman Papillomavirus (HPV) minor capsid protein, L2. Such recombinantVLPs serve as a prophylactic vaccine to prevent infection by diverse HPVstrains.

The vaccines described below induced high titer antibody responsesagainst L2 and protected against HPV challenge in a mouse model ofinfection. Similar techniques could also be used to construct MS2 VLPsthat display L2 peptides.

The Plasmids pP7K and p2P7K32.

Overview of Plasmid Construction.

Two general kinds of plasmid were constructed for the synthesis of PP7coat protein in E coli (see FIGS. 1 and 2). The first (pP7K and p2P7K32)expresses coat protein from the lac promoter and is used (in combinationwith pRZP7—see below) to assay for coat protein's tolerance of peptideinsertions using translational repressor and VLP assembly assays. Thesecond plasmid type (pETP7K and pET2P7K32) expresses the protein fromthe T7 promoter and transcription terminator. These plasmids producelarge amounts of coat protein that assembles correctly into a VLP. Theyalso produce coat-specific mRNA with discrete 5′- and 3′-termini forencapsidation into VLPs.

Design of the Peptide Insertion Site.

The three-dimensional structure of the PP7 capsid shows that it iscomprised of a coat protein whose tertiary structure closely mimics thatof MS2, even though the amino acid sequences of the two proteins showonly about 12% sequence identity (47). The PP7 protein possesses anAB-loop into which peptides may be inserted following a scheme similarto the one we described previously for MS2 (38). As in the MS2 case,mutation of the PP7 coat sequence to contain a site for the restrictionendonuclease KpnI facilitates insertion of foreign sequences in theplasmids called pP7K and pETP7K (FIG. 1). This modification resulted inthe amino acid substitution (E11T) shown in FIG. 3. This substitutionwas well tolerated, since the mutant coat protein represses translationand assembles correctly into a VLP. Again following the MS2 example, itwas assumed that the folding of a single chain dimer version of PP7 coatprotein would be more resistant to AB-loop insertions than theconventional dimer. Its construction was described previously (6). Thesingle-chain dimer was modified to contain a KpnI site only in thedownstream copy of the coding sequence, producing p2P7K32 and pET2P7K32(FIG. 1). In this design, peptides can be inserted at amino acid 11, butit should be noted that other specific insertion sites are possible,including in the amino terminus, in the carboxy terminus or anywherewithin the AB-loop of the coat polypeptide, preferably at amino acids8-11 of the AB loop. Insertion or replacement of amino acids within theAB-loop (or the amino acid or carboxy terminus of the coat polypeptide,preferably within the downstream subunit of the coat polypeptide) may beused to accommodate the L2 peptide.

Example 1 Design of L2-Displaying PP7 VLPs

The construction of the expression plasmids p2P7K32 and pET2P7K32 havebeen described above. As explained, these plasmids code for theexpression of a version of PP7 coat protein in which two copies of coatprotein are genetically fused into a “single-chain” dimer. p2P7K32 andpET2P7K32 also contain a unique KpnI sites that allow for geneticinsertion of sequences at amino acid 11 of the downstream copy of coat.To create the VLPs that display L2 peptides we designed PCR primers(shown in FIG. 2) that allowed us to clone L2-derived sequences into theAB-loop of PP7 coat. These sequences represented L2 amino acids 17-31from different HPV isolates, including HPV16 (QLYKTCKQAGTCPPD) SEQ IDNo. 15, HPV45 (DLYRTCKQSGTCPPD) SEQ ID No. 25, and HPV58(QLYQTCKASGTCPPD) SEQ ID No. 19. This strategy was used to insert thecorresponding L2 amino acids from other HPV types (shown in FIG. 2) intothe PP7 single-chain dimer. The sequence of this region of L2 isrelatively conserved across diverse HPV isolates (FIG. 4).

The functionality of coat protein encoded by the resulting plasmids(including, for example, p2P7-16L2 p2P7-45L2, and p2P7-58L2) was testedby two assays. First, we assessed the translational repression activityof recombinant PP7 coat proteins. PP7 coat normally functions as atranslational repressor, shutting off synthesis of the viral replicaseby binding to a specific RNA hairpin structure containing itsribosome-binding site (the translational operator). We describedpreviously the construction of pRZP7, a plasmid that fuses the PP7translational operator to the E. coli lacZ gene, thus placingβ-galactosidase synthesis under control of coat protein's translationalrepressor activity (28). Because it confers resistance to a differentantibiotic (chloramphenicol), and because it comes from a differentincompatibility group (i.e. it uses the p15A replication origin), it caneasily be maintained in the same E. coli strain as p2P7K32, whichconfers resistance to ampicillin and uses a colE1 origin. The expressionof PP7 coat protein from p2P7K32 represses translation ofβ-galactosidase expressed from pRZP7. This makes it easy to determinewhether a given peptide insertion has interfered with the ability ofcoat protein to correctly fold, since defective coat proteins give bluecolonies on plates containing the β-galactosidase chromogenic substrateknown as X-gal, whereas a properly functioning coat protein yields whitecolonies. All three recombinant coat proteins produced white colonies,indicating that the L2-recombinant coat proteins were functional.

Second, we assessed the presence of VLPs in lysates of cells expressinga peptide-coat protein recombinant by electrophoresis on agarose gel ofcells lysed by sonication. Ethidium bromide staining detects theRNA-containing VLP, whose presence can be confirmed by western blotanalysis using anti-PP7 serum. Electrophoresis of the VLPs in an agarosegel shows that each construct contains RNA (it stains with ethidiumbromide) and exhibits an altered electrophoretic mobility due to chargedifferences conferred by the inserted peptides (FIG. 5). Thus, all threerecombinant single-chain dimer coat proteins formed VLPs.

Example 2 L2 Peptides Displayed on PP7 VLPs are Displayed to the ImmuneSystem and are Immunogenic

Overview.

In order to demonstrate that L2 peptides inserted into the PP7 AB-loopwere indeed displayed on the surface of VLPs, we assessed the ability ofa monoclonal antibody (mAb) RG-1; (20)) specific for the HPV16 L2sequence to bind to recombinant 16L2-PP7 VLPs by ELISA. As shown in FIG.6a , mAb RG-1 bound to 16L2-VLPs, but not to wild-type PP7 VLPs or PP7VLPs that were modified to display the FLAG epitope (FLAG-VLPs).Moreover, as shown in FIG. 6b , mAb RG-1 bound to all eight of theL2-VLPs we produced, but not to wild-type PP7 VLPs.

To test the immunogenicity of the VLPs, groups of three to nine micewere immunized with L2 displaying-VLPs or wild-type PP7 VLPs byintramuscular injection. Groups of three to nine mice were immunizedintramuscularly with 10 μg of VLPs plus incomplete Freunds Adjuvant(IFA). All mice were boosted with the same amount of VLPs two weekslater. Sera were collected before each inoculation and weekly for threeto four weeks after the boost. Sera from the mice were tested, byend-point dilution ELISA, for IgG antibodies specific for synthetic L2peptides representing HPV1, 5, 6, 16, or 18 (FIG. 7). Mice immunizedwith 1L2-, 5L2-, 6L2-, 11L2-, 16L2-, 18L2-, 45L2-, and 58L2-VLPsgenerated high-titer (geometric mean titer typically >10⁴) IgG responsesagainst the corresponding peptide whereas no antibodies were detected incontrol mice. Thus, L2 peptides displayed on the surface of PP7single-chain dimer VLPs display the high immunogenicity that ischaracteristic of other VLP-displayed antigens.

Example 3 PP7 VLPs Displaying a HPV16 L2 Peptide can Induce NeutralizingAntibodies that Protect Mice from Homologous and Heterologous GenitalHPV Pseudovirus Challenge

Overview.

The 16L2-VLP vaccine we designed contains amino acids 17-31 from HPV16L2, a region shown to contain one or more highly cross-reactiveneutralizing epitopes (1, 20), suggesting that the 16L2 VLPs couldpotentially protect against HPV challenge. We demonstrated that16L2-VLPs could protect mice from HPV challenge using a HPVpseudovirus/mouse genital challenge model, first reported by Roberts andcolleagues (40).

Additional Description.

The 16L2-VLP vaccine we designed contains amino acids 17-31 from HPV16L2, a region shown to contain one or more highly cross-reactiveneutralizing epitopes (1, 20), suggesting that the 16L2 VLPs couldpotentially protect against HPV challenge. We assessed whether 16L2-VLPscould protect mice from HPV challenge using a HPV pseudovirus/mousegenital challenge model, first reported by Roberts and colleagues (40).Groups of five Balb/c mice were given two intramuscular injections ofHPV16 L1-VLPs, wild type PP7 VLPs, or 16L2-VLPs, or adjuvant (IFA)alone, and then, three weeks after the boost, challenged intravaginallywith a high dose (˜10⁸ IU) of HPV pseudovirus carrying a luciferasereporter. As a negative control, mice were mock-challenged with PBS.Infection was detected as a bioluminescent signal two days after theadministration of pseudovirions, immediately after intravaginalinstillation of the challenged mice with the reporter substrate,luciferin.

As shown in FIG. 8, mice immunized with 16L2-VLPs were strongly (˜90%)protected from infection with the homologous pseudovirus, HPV16, whereasmice immunized with wild-type PP7 VLPs were not protected. We alsotested whether vaccination with 16L2-VLPs could protect mice fromgenital infection with a heterologous HPV type. We chose HPV45pseudovirus because it is not closely related to HPV16 and because itsL2(17-31) sequence varies from the HPV16 sequence at three of thefifteen amino acid positions. Immunization with HPV16 L1 VLPs did notprotect mice from HPV45 challenge. However, 16L2-VLP-immunized mice wereprotected (˜83%) from genital infection with HPV45 pseudovirus. Thus,16L2-VLPs have potential as a pan-HPV vaccine.

Example 4 A Mixture of PP7 VLPs Displaying HPV L2 Peptides can InduceAntibodies that Protect Mice from Homologous and Heterologous GenitalHPV Pseudovirus Challenge

We also tested the immunogenicity of a combination vaccine consisting ofall eight L2-PP7 VLPs that were constructed. Groups of mice wereimmunized with a mixture of equal amounts of 1L2-VLPs, 5L2-VLPs,6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. Micewere immunized three times at two-week intervals with a 10 μg dose andwithout exogenous adjuvant. Sera were collected before each inoculationand weekly for three to four weeks after the boost. Sera from the micewere tested, by end-point dilution ELISA, for IgG antibodies specificfor synthetic L2 peptides representing HPV1, 5, 6, 16, or 18 (FIG. 9).Mice immunized with the mixture of L2-VLPs produced high-titerantibodies reactive with peptides representing 1L2, 5L2, 6L2, 11L2,16L2, and 18L2. Thus, a mixture of L2-VLPs displayed on the surface ofPP7 single-chain dimer VLPs is also highly immunogenic.

We assessed whether the mixed L2-VLP vaccination could protect mice fromHPV challenge using the HPV pseudovirus/mouse genital challenge modeldescribed above. Following immunization with the mixed L2-VLPs (asdescribed above) or, as a negative control, wild-type PP7 VLPs, micewere challenged intravaginally with a high dose (10⁷-10⁸ IU) of HPV5, 6,16, 18, 31, 45, 52, or 58 pseudovirus carrying a luciferase reporter. Asshown in FIG. 10, immunization with mixed PP7 L2-VLPs protected micefrom HPV5 pseudovirus infection (98.2% reduction in signal), HPV6pseudovirus infection (98.6% reduction in signal), HPV16 pseudovirusinfection (99.7% reduction in signal), HPV18 pseudovirus infection(99.1% reduction in signal), HPV31 pseudovirus infection (99.9%reduction), HPV45 pseudovirus infection (99.2% reduction), HPV52pseudovirus infection (98.5% reduction in signal), and HPV58 pseudovirusinfection (93.1% reduction in signal). Thus, mixed L2-VLPs also havepotential as a pan-HPV vaccine.

Example 5 Other Regions of HPV L2 can be Displayed on the Surface ofBacteriophage VLPs and are Immunogenic

Using the methods described elsewhere in this application, we generatedrecombinant PP7 VLPs that display HPV16 L2 amino acids 35-50 (amino acidsequence: KVEGKTIADQILQYGS) SEQ ID No. 36 and amino acids 51-65(sequence: MGVFFGGLGIGTGSG), SEQ ID No. 37. These VLPs were used toimmunize mice, and, following two immunizations, antibodies against asynthetic peptides representing A) HPV16 L2 amino acids 34-52 or B)HPV16 L2 amino acids 49-71 were measured by end-point dilution ELISA. Asshown in FIG. 11, both recombinant VLPs induced high-titer IgGantibodies that recognized the L2 peptides.

REFERENCES

-   1. Alphs, H. H., R. Gambhira, B. Karanam, J. N. Roberts, S.    Jagu, J. T. Schiller, W. Zeng, D. C. Jackson, and R. B. Roden. 2008.    Protection against heterologous human papillomavirus challenge by a    synthetic lipopeptide vaccine containing a broadly    cross-neutralizing epitope of L2. Proc Natl Acad Sci USA 105:5850-5.-   2. Bachmann, M. F., U. H. Rohrer, T. M. Kundig, K. Burki, H.    Hengartner, and R. M. Zinkernagel. 1993. The influence of antigen    organization on B cell responsiveness. Science 262:1448-1451.-   3. Bachmann, M. F., and R. M. Zinkernagel. 1997. Neutralizing    antiviral B cell responses. Annu Rev Immunol 15:235-70.-   4. Brunswick, M., F. D. Finkelman, P. F. Highet, J. K. Inman, H. M.    Dintzis, and J. J. Mond. 1988. Picogram quantities of anti-Ig    antibodies coupled to dextran induce B cell proliferation. J Immunol    140:3364-72.-   5. Buck, C. B., N. Cheng, C. D. Thompson, D. R. Lowy, A. C.    Steven, J. T. Schiller, and B. L. Trus. 2008. Arrangement of L2    within the papillomavirus capsid. J Virol 82:5190-7.-   6. Caldeira, J. C., and D. S. Peabody. 2007. Stability and assembly    in vitro of bacteriophage PP7 virus-like particles. J    Nanobiotechnology 5:10.-   7. Campo, M. S., G. J. Grindlay, B. W. O'Neil, L. M.    Chandrachud, G. M. McGarvie, and W. F. Jarrett. 1993. Prophylactic    and therapeutic vaccination against a mucosal papillomavirus. J Gen    Virol.-   8. Chackerian, B., L. Briglio, P. S. Albert, D. R. Lowy, and J. T.    Schiller. 2004. Induction of autoantibodies to CCR5 in macaques and    subsequent effects upon challenge with an R5-tropic simian/human    immunodeficiency virus. J Virol 78:4037-47.-   9. Chackerian, B., M. R. Durfee, and J. T. Schiller. 2008.    Virus-like display of a neo-self antigen reverses B cell anergy in a    B cell receptor transgenic mouse model. J Immunol 180:5816-25.-   10. Chackerian, B., D. R. Lowy, and J. T. Schiller. 2001.    Conjugation of a self-antigen to papillomavirus-like particles    allows for efficient induction of protective autoantibodies. J Clin    Invest 108:415-23.-   11. Chackerian, B., D. R. Lowy, and J. T. Schiller. 1999. Induction    of autoantibodies to mouse CCR5 with recombinant papillomavirus    particles. Proc. Natl. Acad. Sci. USA 96:2373-2378.-   12. Chackerian, B., M. Rangel, Z. Hunter, and D. S. Peabody. 2006.    Virus and virus-like particle-based immunogens for Alzheimer's    disease induce antibody responses against amyloid-beta without    concomitant T cell responses. Vaccine 24:6321-31.-   13. Christensen, N. D., J. W. Kreider, N. C. Kan, and S. L.    DiAngelo. 1991. The open reading frame L2 of cottontail rabbit    papillomavirus contains antibody-inducing neutralizing epitopes.    Virology 181:572-9.-   14. Cornuz, J. S. Zwahlen, W. F. Jungi, J. Osterwalder, K.    Klingler, G. van Melle, Y. Bangala, I. Guessous, P. Muller, J.    Willers, P. Maurer, M. F. Bachmann, and T. Cerny. 2008. A vaccine    against nicotine for smoking cessation: a randomized controlled    trial. PLoS ONE 3:e2547.-   15. Day, P. M., R. Gambhira, R. B. Roden, D. R. Lowy, and J. T.    Schiller, 2008. Mechanisms of human papillomavirus type 16    neutralization by 12 cross-neutralizing and 11 type-specific    antibodies. J Virol 82:4638-46.-   16. Dintzis, H. M., R. Z. Dintzis, and B. Vogelstein. 1976.    Molecular determinants of immunogenicity: the immunon model of    immune response. Proc Natl Acad Sci USA 73:3671-5.-   17. Dintzis, R. Z., M. H. Middleton, and H. M. Dintzis. 1985.    Inhibition of anti-DNP antibody formation by high doses of    DNP-polyacrylamide molecules; effects of hapten density and hapten    valence. J Immunol 135:423-7.-   18. Fehr, T., D. Skrastina, P. Pumpens, and R. M. Zinkernagel. 1998.    T cell-independent type I antibody response against B cell epitopes    expressed repetitively on recombinant virus particles. Proc Natl    Acad Sci USA 95:9477-81.-   19. Gambhira, R., S. Jagu, B. Karanam, P. E. Gravitt, T. D.    Culp, N. D. Christensen, and R. B. Roden. 2007. Protection of    rabbits against challenge with rabbit papillomaviruses by    immunization with the N terminus of human papillomavirus type 16    minor capsid antigen L2. J Virol 81:11585-92.-   20. Gambhira, R., B. Karanam, S. Jagu, J. N. Roberts, C. B. Buck, I.    Bossis, H. Alphs, T. Culp, N. D. Christensen, and R. B. Roden. 2007.    A protective and broadly cross-neutralizing epitope of human    papillomavirus L2. J Virol 81:13927-31.-   21. Ghim, S. J., A. B. Jenson, and R. Schlegel. 1992. HPV-1 L1    protein expressed in cos cells displays conformational epitopes    found on intact virions. Virology 190:548-52.-   22. Harro, C. D., Y. Y. Pang, R. B. Roden, A. Hildesheim, Z.    Wang, M. J. Reynolds, T. C. Mast, R. Robinson, B. R. Murphy, R. A.    Karron, J. Dillner, J. T. Schiller, and D. R. Lowy. 2001. Safety and    immunogenicity trial in adult volunteers of a human papillomavirus    16 L1 virus-like particle vaccine. J Natl Cancer Inst 93:284-92.-   23. Kawana, K., Y. Kawana, H. Yoshikawa, Y. Taketani, K. Yoshiike,    and T. Kanda. 2001. Nasal immunization of mice with peptide having a    cross-neutralization epitope on minor capsid protein L2 of human    papillomavirus type 16 elicit systemic and mucosal antibodies.    Vaccine 19:1496-502.-   24. Kirnbauer, R., F. Booy, N. Cheng, D. R. Lowy, and J. T.    Schiller. 1992. Papillomavirus L1 major capsid protein    self-assembles into virus-like particles that are highly    immunogenic. Proc Natl Acad Sci USA 89:12180-12184.-   25. Kirnbauer, R., J. Taub, H. Greenstone, R. B. S. Roden, M.    Durst, L. Gissmann, D. R. Lowy, and J. T. Schiller. 1993. Efficient    self-assembly of human papillomavirus type 16 L1 and L1-L2 into    virus-like particles. J Virol 67:6929-6936.-   26. Koutsky, L. A., K. A. Ault, C. M. Wheeler, D. R. Brown, E.    Barr, F. B. Alvarez, L. M. Chiacchierini, and K. U. Jansen. 2002. A    controlled trial of a human papillomavirus type 16 vaccine. N Engl J    Med 347:1645-51.-   27. Li, Q., C. Cao, B. Chackerian, J. Schiller, M. Gordon, K. E.    Ugen, and D. Morgan. 2004. Overcoming antigen masking of    anti-amyloidbeta antibodies reveals breaking of B cell tolerance by    virus-like particles in amyloidbeta immunized amyloid precursor    protein transgenic mice. BMC Neurosci 5:21.-   28. Lim, F., T. P. Downey, and D. S. Peabody. 2001. Translational    repression and specific RNA binding by the coat protein of the    Pseudomonas phage PP7. J Biol Chem 276:22507-13.-   29. Lim, F., and D. S. Peabody. 2002. RNA recognition site of PP7    coat protein. Nucleic Acids Res 30:4138-44.-   30. Lin, Y.-L., L. A. Borenstein, R. Selvakumar, R. Ahmed, and F. O.    Wettstein. 1992. Effective vaccination against papilloma development    by immunization with L1 or L2 structural protein of cottontail    rabbit papillomavirus. Virology 187:612-619.-   31. Mao, C., L. A. Koutsky, K. A. Ault, C. M. Wheeler, D. R.    Brown, D. J. Wiley, F. B. Alvarez, O. M. Bautista, K. U. Jansen,    and E. Barr. 2006. Efficacy of human papillomavirus-16 vaccine to    prevent cervical intraepithelial neoplasia: a randomized controlled    trial. Obstet Gynecol 107:18-27.-   32. Milich, D. R., M. Chen, F. Schodel, D. L. Peterson, J. E. Jones,    and J. L. Hughes. 1997. Role of B cells in antigen presentation of    the hepatitis B core. Proc Natl Acad Sci USA 94:14648-53.-   33. Munoz, N., F. X. Bosch, S. de Sanjose, R. Herrero, X.    Castellsague, K. V. Shah, P. J. Snijders, and C. J. Meijer. 2003.    Epidemiologic classification of human papillomavirus types    associated with cervical cancer. N Engl J Med 348:518-27.-   34. Parkin, D. M., and F. Bray. 2006. Chapter 2: The burden of    HPV-related cancers. Vaccine 24 Suppl 3:S3/11-25.-   35. Pastrana, D. V., C. B. Buck, Y. Y. Pang, C. D. Thompson, P. E.    Castle, P. C. FitzGerald, S. Kruger Kjaer, D. R. Lowy, and J. T.    Schiller. 2004. Reactivity of human sera in a sensitive,    high-throughput pseudovirus-based papillomavirus neutralization    assay for HPV16 and HPV18. Virology 321:205-16.-   36. Pastrana, D. V., R. Gambhira, C. B. Buck, Y. Y. Pang, C. D.    Thompson, T. D. Culp, N. D. Christensen, D. R. Lowy, J. T. Schiller,    and R. B. Roden. 2005. Cross-neutralization of cutaneous and mucosal    Papillomavirus types with anti-sera to the amino terminus of L2.    Virology 337:365-72.-   37. Peabody, D. S. 1990. Translational repression by bacteriophage    MS2 coat protein expressed from a plasmid. A system for genetic    analysis of a protein-RNA interaction. J Biol Chem 265:5684-9.-   38. Peabody, D. S., B. Manifold-Wheeler, A. Medford, S. K.    Jordan, J. do Carmo Caldeira, and B. Chackerian. 2008. Immunogenic    display of diverse peptides on virus-like particles of RNA phage    MS2. J Mol Biol 380:252-63.-   39. Richards, R. M., D. R. Lowy, J. T. Schiller, and P. M.    Day. 2006. Cleavage of the papillomavirus minor capsid protein, L2,    at a furin consensus site is necessary for infection. Proc Natl Acad    Sci USA 103:1522-7.-   40. Roberts, J. N., C. B. Buck, C. D. Thompson, R. Kines, M.    Bernardo, P. L. Choyke, D. R. Lowy, and J. T. Schiller. 2007.    Genital transmission of HPV in a mouse model is potentiated by    nonoxynol-9 and inhibited by carrageenan. Nat Med 13:857-61.-   41. Roden, R. B., W. I. Yutzy, R. Fallon, S. Inglis, D. R. Lowy,    and J. T. Schiller. 2000. Minor capsid protein of human genital    papillomaviruses contains subdominant, cross-neutralizing epitopes.    Virology 270:254-7.-   42. Roden, R. B. S., N. L. Hubbert, R. Kirnbauer, N. D.    Christensen, D. R. Lowy, and J. T. Schiller. 1996. Assessment of the    serological relatedness of genital human papillomaviruses by    hemagglutination inhibition. J. Virol 70:3298-3301.-   43. Rose, R. C., W. Bonnez, R. C. Reichman, and R. L. Garcea. 1993.    Expression of human papillomavirus type 11 L1 protein in insect    cells: in vivo and in vitro assembly of viruslike particles. J Virol    67:1936-44.-   44. Schiller, J. T., and D. R. Lowy. 2001. Papillomavirus-like    particle based vaccines: cervical cancer and beyond. Expert Opin    Biol Ther 1:571-81.-   45. Selinka, H. C., T. Giroglou, T. Nowak, N. D. Christensen, and M.    Sapp. 2003. Further evidence that papillomavirus capsids exist in    two distinct conformations. J Virol 77:12961-7.-   46. Stanley, M., D. R. Lowy, and I. Frazer. 2006. Chapter 12:    Prophylactic HPV vaccines: underlying mechanisms. Vaccine 24 Suppl    3:S3/106-13.-   47. Tars, K., K. Fridborg, M. Bundule, and L. Liljas. 2000. The    three-dimensional structure of bacteriophage PP7 from Pseudomonas    aeruginosa at 3.7-A resolution. Virology 272:331-7.-   48. Thyagarajan, R., N. Arunkumar, and W. Song. 2003. Polyvalent    antigens stabilize B cell antigen receptor surface signaling    microdomains. J Immunol 170:6099-106.-   49. Tissot, A. C., P. Maurer, J. Nussberger, R. Sabat, T.    Pfister, S. Ignatenko, H. D. Volk, H. Stocker, P. Muller, G. T.    Jennings, F. Wagner, and M. F. Bachmann. 2008. Effect of    immunisation against angiotensin II with CYT006-AngQb on ambulatory    blood pressure: a double-blind, randomised, placebo-controlled phase    IIa study. Lancet 371:821-7.-   50. Zhang, L. F., J. Zhou, S. Chen, L. L. Cal, Q. Y. Bao, F. Y.    Zheng, J. Q. Lu, J. Padmanabha, K. Hengst, K. Malcolm, and I. H.    Frazer. 2000. HPV6b virus like particles are potent immunogens    without adjuvant in man. Vaccine 18:1051-8.-   51. Zhou, J., X. Y. Sun, D. J. Stenzel, and I. H. Frazer. 1991.    Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in    epithelial cells is sufficient for assembly of HPV virion-like    particles. Virology 185:251-257.

What is claimed is:
 1. A PP7 RNA bacteriophage virus-like particlecomprising a PP7 RNA bacteriophage single chain coat polypeptide dimerhaving an immunogenic papillomavirus (PV) L2 peptide insertion in the ABloop, the peptide insertion consisting essentially of the amino acidsequence corresponding to amino acids 17-31 of HPV16 L2 protein, whichis displayed on the surface of the virus-like particle.
 2. The RNAbacteriophage virus-like particle of claim 1, wherein the L2 peptideinsertion further comprises a second L2 peptide sequence from a secondPV.
 3. The RNA bacteriophage virus-like particle of claim 1, furthercomprising a second RNA bacteriophage single chain coat polypeptidedimer having a second immunogenic papillomavirus (PV) L2 peptideinsertion.
 4. The RNA bacteriophage virus-like particle of claim 1,wherein the second L2 peptide sequence corresponds to amino acids 17-31of HPV16 is selected from HPV1, HPV5, HPV8, HPV35, HPV31, HPV33, HPV58,HPV52, HPV73, HPV6, HPV11, HPV18, HPV45, HPV39, HPV68, HPV59, HPV51,HPV56, HPV 66, HPV2, CPRV and BPV1.
 5. An immunogenic compositioncomprising a bacteriophage virus-like particle of claim
 1. 6. Aviral-like particle comprising a PP7 bacteriophage single chain coatpolypeptide dimer having an HPV L2 peptide insertion consistingessentially of amino acids of SEQ ID NO:24, wherein the HPV L2 peptideis displayed on the virus-like particle.
 7. A method for inducing animmune response to HPV in a subject comprising administering to thesubject an RNA bacteriophage virus-like particle of claim 1, wherein thecomposition induces a HPV-immunogenic response in the patient.
 8. TheRNA bacteriophage virus-like particle of claim 1, wherein theimmunogenic papillomavirus (PV) L2 peptide insertion has an amino acidsequence that is identical to amino acids 17-31 of HPV16 L2 protein.